Bioactive lipids such as prostaglandin (PG), thromboxane (TX), leukotriene (LT), and sphingosine-1-phosphate play a critical physiological role in the etiology of various disorders. (Wymann, M P and Schneiter R, Nat. Rev. Mol. Cell. Biol. 9(2):162-76 (2008)). During inflammation, cellular phospholipases, especially phospholipases A2 and C, are activated and degrade cell membrane phospholipids to arachidonic acid (AA). AA is metabolized by two major routes, the cyclooxygenase (COX) and lipooxygenase (LO) pathways. The COX pathway produces prostaglandins (PGD2, PGE2, PGF2α, prostacyclin or PGI2, and thromboxane A2 or TXA2). The LO pathway has two branches; the 5-LO pathway produces leukotrienes (e.g., LTA4, LTB4, LTC4, LTD4, LTE4, and LTF4) and the 15-LO pathway produces lipoxins (e.g., LXA4, LXB4). Prostanoids such as prostaglandin (PG), thromboxane (TX) and leukotriene (LT) have various physiological activities for maintaining local homeostasis in the body (The Pharmacological Basis of Therapeutics, Gilman, et al., eds., 7th Ed., p. 660, Macmillan Publishing Co., New York (1985)). The products of COX, PG G2/PG H2, are converted to specific PGs by the actions of tissue specific isomerases to yield PGI2, TXA2, PGD2, PGE2, and PGF2α. The biological functions of PGs are mediated by tissue-specific cell surface rhodopsin-like seven transmembrane spanning G protein-coupled receptors (GPCRs). The precise physiological/pathological role of each PG is determined by the cellular context, receptor expression profile, ligand affinity, and differential coupling to signal transduction pathways (Haluska et al., Annu. Rev. Pharm. Tox. 10:213 (1989); Prostanoids and their Receptors. In Comprehensive Medicinal Chemistry, p. 643, Pergamon Press, Oxford (1990)). PGs play a wide variety of physiological roles in the regulation of modulation of vasomotricity, the sleep/wake cycle, intestinal secretion, lipolysis, glomerular filtration, mast cell degranulation, neurotransmission, platelet aggregation, leuteolysis, myometrial contraction and labor, inflammation and arthritis, patent ductus arteriosus, cell growth and differentiation, and immune responses generally. Patho-physiologically, PGs have been implicated in a variety of diseases including pain and inflammation, cancer, neurological diseases, cardiovascular diseases, and hypertension.
Prostaglandin E2 (PGE2) is a member of the prostanoid family. PGE2 participates widely in the contraction and relaxation of the gastrointestinal tract, secretion of gastric acid, relaxation of smooth muscle, and release of neurotransmitters. Four subtype receptors for PGE2 have been identified, including EP1, EP2, EP3, and EP4 (Negishi, M. et al., J. Lipid Mediators Cell Signalling, 12:379-391 (1995)), each of which is involved in a different signal transduction pathway.
PGE2 is the main product of the COX pathway of AA metabolism. It is the major PG synthesized in the joints and plays an important role in inflammation and the pathogenesis of arthritis. Five PGE2 synthases have been identified. (Smith W L, Am. J. Physiol. 263(2 Pt 2):F181-91 (1992)). Of these five, membrane PGE synthase (mPGES)-1 appears to be the key PGE2 convertase enzyme responsible for PGE2 production. MPGES-1 displays the highest catalytic activity relative to other PGE synthases and functions in conjunction with COX-1 and/or COX-2, to convert PGH2 to PGE2. Studies using mPGES-1 KO mice (Kamei, D., et al., J. Biol. Chem., 279(32):33684-95 (2004); Trebino, C. E., et al., Proc. Natl. Acad. Sci. USA 100(15):9044-9 (2003), specific PGE2 receptor isoform KO mice (McCoy, J. M., et al. J. Clin. Invest., 110(5):651-8 (2002); Majima, M., et al. Trends Pharmacol. Sci., 24(10):524-9 (2003); and Amano, H., et al., J. Exp. Med., 197(2):221-32 (2003); and anti-PGE2 specific antibodies (Portanova, J. P., et al., J. Exp. Med., 184(3):883-91 (1996); Zhang, Y., et al., J. Pharmacol. Exp. Ther., 283(3):1069-75 (1997) suggest that PGE2 plays a major role in animal models of rheumatoid arthritis (RA), pain and inflammation and cancer development. In the absence of mPGES-1, levels of COX-1, COX-2, and other PGE2 synthases remain relatively unaltered. The mPGES-1 KO mice are viable, fertile, and develop normally compared to wild type mice. However, they display a drastic reduction in both basal levels of PGE2 production as well as in PGE2 production from macrophages following challenge with various inflammatory stimuli. In addition, production of TXA2 is increased. The mPGES-1 KO mice show reduced incidence and severity of arthritis development and show resistance to pain and inflammation in various models. Several laboratories have independently generated various EP receptor isoform KO mice. These mice are viable, fertile and develop normally. Studies using specific EP isoform KO mice demonstrate that the various functions of PGE2 are mediated via specific EP isoforms. For example, the lack of EP4 isoform clearly affects the severity of arthritis development in mice, whereas the lack of EP3 influences tumor development and progression by modulating VEGF production by stromal cells and angiogenesis.
Defects in the biosynthesis and metabolism of prostaglandins are now believed to play an important part in the etiology of autoimmune and inflammatory disorders. For example, the synovial tissues from patients suffering from rheumatoid arthritis produce larger amounts of PGE2 and prostaglandin F2α, (PGF2α) compared to the synovial tissues from unaffected subjects (Blotman, F., et al., Rev. Rhum. Mal. Osteoartic, 46(4):243-7 (1979)). Similarly, an increased synthesis of PGE2 and PGF2α occurs in patients exhibiting systemic and gastrointestinal symptoms secondary to food intolerance. Thus, migraine headaches secondary to the ingestion of certain foods could be the result of an increased synthesis of 2-series prostaglandins. Multiple sclerosis is also associated with an imbalance in the normal levels of the prostaglandins, PGE1 and PGE2. Many aspects of reproduction, for example, fertility, pregnancy and labor, may be regulated by prostaglandins. Prostaglandins also play a major role in reproductive physiology. Excessive prostaglandin synthesis causes dysmenorrhea and parturition, which may be induced by administering prostaglandins intravenously or by insertion of a prostaglandin pessary. (Wang L. et al., Occup. Environ. Med. 61(12):1021-1026 (2004)). Excessive synthesis of PGE2 also plays a major role in disorders of reproduction, such as infertility, repeated miscarriage, preeclampsia, and eclampsia. A need therefore exists for antibodies specific to PGE2 that block or modulate its biological functions, which may be used to prevent and treat the diseases associated with excess production of PGE2 as well as diagnostic purposes.
The generation of a highly specific, high affinity (KD is about 300 pM) anti-PGE2 mAb, 2B5, has been reported. (Mnich S J, et al. J. Immunol. 155(9):4437-44 (1995)). The efficacy of 2B5 relative to indomethacin, a COX-1,2 inhibitor, was determined in animal models of pain and inflammation in mice and adjuvant-induced arthritis in rats. (Portanova J P et al., J. Exp. Med. 184(3):883-91 (1996)). These studies clearly showed that 2B5 was as effective as indomethacin in reducing pain and inflammation as well as the severity of arthritis, suggesting that PGE2 is a key participant in the COX-1,2 pathway of AA metabolism in these animal models.
Inhibition of pan-PG production by COX-inhibitors has been a well-established therapeutic strategy for decades. Two isoforms of COX, COX-1 and COX-2, are known, each of which are encoded by a distinct gene. The two isoforms carry out essentially the same catalytic reaction and have similar tertiary structures (Garavito R M, et al., Annu. Rev. Biophys. Biomol. Struct. 32:183-206 (2003)). COX-1 is constitutively expressed in nearly all tissues and is believed to be largely responsible for the normal “house keeping” functions, such as gastric cytoprotection and homeostasis. COX-2, by contrast, is constitutively expressed in particular tissues, and is highly inducible at sites of inflammation and cancer. Thus, COX-2-mediated PG production is thought to play an important role at the site of inflammation and cancer. The traditional non-steroidal anti-inflammatory drugs (NSAIDs), e.g., aspirin, indomethacin, ibuprofen) inhibit both COX isoforms. These compounds are the most widely used drugs for pain, rheumatoid arthritis (RA), osteoarthritis (OA), and cardiovascular diseases and now are under consideration for the prevention of colon cancer and AD. The main liabilities of traditional NSAIDs are gastric and renal adverse events, in high-risk populations, which are believed to be due to inhibition of COX-1. Therefore, the second generation of NSAIDs, the COX-2 selective inhibitors (e.g., celecoxib, Celebrex™; rofecoxib, Vioxx™; valdecoxib, Bextra™), are believed to have a better therapeutic profile. This assumption has resulted in their widespread use for pain, RA, and OA. Since the approval of the first COX-2 inhibitor in 1999 the combined sales of COX-2 inhibitors in 2004 was approximately US $ 5 billion. However, recently some COX-2 selective inhibitors were taken off the market, and are under FDA review, due to cardiovascular side-effects in high risk patients for certain COX-2 inhibitors. The liabilities associated with COX inhibitors probably arose due to their ability to inhibit all PGs, and in particular due to their ability to differentially interfere with PGI2 and TXA2 production, both of which play an important role in maintaining cardiovascular homeostasis (Martinez-González J. et al., Curr. Pharm. Des. 13(22):2215-2227 (2007)). The inhibition of COX may make more AA available to the LO pathways, thus increasing the production of leukotrienes and lipoxins, which may contribute to COX inhibition-associated adverse effects. Recent studies using COX-1 and/or COX-2 knockout mice and COX-1 and COX-2 specific inhibitors also suggest that assumptions concerning the physiological roles of the two COX-isoforms may not be correct. (Loftin, C. D., et al. Prostaglandins Other Lipid Mediat. 68-69:177-85 (2002)). These studies suggest that both COX-1 and COX-2 play an important role in supplying PGs to maintain tissue homeostasis and both isoforms may contribute to disease development, such as pain, inflammation and cancer. Therefore, blocking detrimental PGE2 downstream of COX-1 and COX-2 pathway with a specific antibody appears to be an attractive approach for the treatment of certain human diseases.
Another example of an important bioactive prostaglandin is PGD2. PGD2 is the major cyclooxygenase product of arachidonic acid produced from mast cells on immunological challenge (Lewis, et al., J. Immunol. 129:1627-1631 (1982)). Activated mast cells, a major source of PGD2, are one of the key players in driving the allergic response in conditions such as asthma, allergic rhinitis, allergic conjunctivitis, allergic dermatitis and other diseases (Brightling, et al., Clin. Exp. Allergy 33:550-556 (2003)). Recent studies have shown that PGD2 exerts its effects through two different G-protein-coupled receptors (GPCRs), the D-prostanoid receptor (DP) and the chemoattractant receptor-homologous molecule expressed on T helper type-2 cells (CRTH2), expressed in various human tissues. The PGD2/CRTH2 system mediates the chemotaxis of eosinophils, basophils, and Th2 cells, which are involved in the induction of allergic inflammation (Ulven T et al., Curr. Top. Med. Chem. 6(13):1427-1444 (2006)). Many of the actions of PGD2 are mediated through its action on the D-type prostaglandin (“DP”) receptor, a G protein-coupled receptor expressed on epithelium and smooth muscle. In asthma, the respiratory epithelium has long been recognized as a key source of inflammatory cytokines and chemokines that drive the progression of the disease (Holgate, et al., Am. J. Respir. Crit. Care Med. 162:113-117 (2000)). In an experimental murine model of asthma, the DP receptor is dramatically up-regulated on airway epithelium on antigen challenge (Matsuoka, et al., Science 287:2013-2017 (2000)). The DP receptor is involved in human allergic rhinitis, a frequent allergic disease that is characterized by the symptoms of sneezing, itching, rhinorea and nasal congestion. DP antagonists have been shown to be effective at alleviating the symptoms of allergic rhinitis in multiple species, and more specifically have been shown to inhibit the antigen-induced nasal congestion, the most manifest symptom of allergic rhinitis (Jones, et al., Am. J. Resp. Crit. Care Med. 167:A218 (2003); Arimura, et al., S-5751. J. Pharmacol. Exp. Ther. 298(2):411-9 (2001)). DP antagonists are also effective in experimental models of allergic conjunctivitis and allergic dermatitis (Arimura et al., S-5751. J. Pharmacol. Exp. Ther. 298(2):411-9 (2001); Torisu, et al., Bioorg. & Med. Chem. 12:5361-5378 (2004)). A need therefore also exists for antibodies specific to PGD2 and blocking or modulating its biological functions therefore may be used to prevent and treat the diseases associated with excess production of PGD2.
Sphingosine-1-phosphate (S1P) is another example of a bioactive lipid that induces many cellular effects, including those that result in platelet aggregation, cell proliferation, cell morphology, tumor cell invasion, endothelial cell chemotaxis, and endothelial cell in vitro angiogenesis. S1P receptors are therefore good targets for therapeutic applications such as wound healing and tumor growth inhibition. S1P signals cells in part via a set of G protein-coupled receptors named S1P1, S1P2, S1P3, S1P4, and S1P5 (formerly called EDG-1, EDG-5, EDG-3, EDG-6, and EDG-8, respectively). These receptors share 50-55% amino acid and cluster identity with three other receptors (LPA1, LPA2, and LPA3 (formerly EDG-2, EDG-4 and EDG-7)) for the structurally-related lysophosphatidic acid (LPA). (Ishii, I. et al., Mol. Pharmacol. 58(5):895-902 (2000)). A conformational shift is induced in the G-Protein Coupled Receptor (GPCR) when the ligand binds to that receptor, causing GDP to be replaced by GTP on the α-subunit of the associated G-proteins and subsequent release of the G-proteins into the cytoplasm. The α-subunit then dissociates from the βγ-subunit, and each subunit can then associate with effector proteins, which activate second messengers leading to a cellular response. Eventually the GTP on the G-proteins is hydrolyzed to GDP, and the subunits of the G-proteins re-associate with each other and then with the receptor. Amplification plays a major role in the general GPCR pathway. The binding of one ligand to one receptor leads to the activation of many G-proteins, each capable of associating with many effector proteins, leading to an amplified cellular response. S1P receptors make good drug targets, because individual receptors are both tissue- and response-specific. Tissue specificity of the S1P receptors is important, because development of an agonist or antagonist selective for one receptor localizes the cellular response to tissues containing that receptor, limiting unwanted side effects. Response specificity of the S1P receptors is also important because it allows for development of agonists or antagonists that initiate or suppress certain cellular responses without affecting other responses. For example, the response specificity of the S1P receptors could allow for an S1P mimetic that initiates platelet aggregation without affecting cell morphology.
S1P is formed as a metabolite of sphingosine in its reaction with sphingosine kinase, and is abundantly stored in platelet aggregates where high levels of sphingosine kinase exist and sphingosine lyase is absent. S1P is released during platelet aggregation, accumulates in serum and is also found in malignant ascites. S1P biodegradation most likely proceeds via hydrolysis by ectophosphohydrolases, specifically the sphingosine 1-phosphate phosphohydrolases. A need therefore exists for antibodies specific to S1P for modulating its biological functions either by blocking its interaction with receptors or stabilizing S1P and enhancing its biological effects, for use in preventing or treating autoimmune diseases, inflammatory diseases, and cancers.
Due to the role of PGE2 in a variety of human disorders, therapeutic strategies have been designed to inhibit or counteract PGE2 activity. In particular, therapeutic antibodies suitable for delivery to humans that bind to, and neutralize, PGE2 have not been reported. There exists a need in the art for improved antibodies capable of binding and neutralizing PGE2.